The non-pathogenic yeast Starmerella bombicola (formerly known as Candida bombicola), ATCC 22214 (CBS 6009), is commercially applied for the production of sophorolipids (FIG. 1). Ideally, production of sophorolipids involves the presence of an easily metabolized carbohydrate such as glucose and an oil or fatty acid (e.g., vegetable oil) as, respectively, the hydrophilic and hydrophobic carbon source. These can be considered as the conventional carbon sources applied in sophorolipid production.
Common sophorolipids are comprised of a sophorose head group (2-O-β-D-glucopyranosyl-D-glucopyranose) attached to a (sub)terminal hydroxylated C18 or C16 fatty acid by a glycosidic linkage between the anomeric C-atom of the sugar and the hydroxyl group of the fatty acid. Sophorolipids are always comprised of a mixture of structurally related molecules with variation in: 1) degree of fatty acid saturation (saturated, mono-unsaturated or di-unsaturated), 2) presence or absence of acetyl groups at C6′ and/or C6″ atoms, 3) lactonization between the carboxyl end of the fatty acid and either the C4″, C6′ or C6″ atom of the sophorose group resulting in a lactonic sophorolipid or absence of this lactonization resulting in an open or acidic sophorolipid, 4) fatty acid chain length, and 5) (ω) or (ω-1) hydroxylation of the fatty acid (Asmer et al., 1988). Due to this structural variation, sophorolipids show many interesting applications in a wide range of industrial fields (Banat et al., 2010; Franzetti et al., 2010; Kralova and Sjoblom, 2009; and Mulligan, 2009). Since the structural composition of sophorolipids is reflected in the physico-chemical properties, several industries are particularly interested in specific structural variants.
WO 2012/080116 and Saerens et al. (2011b) disclose that yeast strains that are mutated in a gene encoding for an acetyltransferase (at; GenBank accession number HQ670751) are capable of producing a mixture of entirely unacetylated sophorolipids. This acetyltransferase is solely responsible for the acetylation of the sophorolipid molecules at the C6′ and/or C6″ atoms. The unacetylated molecules produced by the knock-out strain display better water solubility and foam-forming capacities.
As explained above, common sophorolipids consist of two glucose molecules forming sophorose with a hydrophobic fatty acid chain attached to it. However, in some exceptional conditions, minor amounts of glycolipids with additional glucose molecules can be detected. The supplementary glucose molecule(s) are found at the other end of the alkyl chain, in this way creating bolaamphiphilic or bolaform molecules, molecules with two hydrophilic parts connected by a hydrophobic spacer. Bolaform surfactants recently attracted a lot of attention due to their unique configuration: a long hydrophobic spacer with hydrophilic groups at both ends. This makes the molecule more water soluble, but still allows formation of micelles, vesicles and other laminar structures. The best known examples of a natural bolaform are the tetraether lipids present in membranes of archaea able to grow under extreme temperatures and salt concentrations. These bolaforms strongly stabilize the membranes under extreme conditions.
The production of bolaform glycolipids appears insufficient and/or expensive. Brakemeier et al. (1995 and 1998) were the first to report on the presence of bolaform sophorolipids when 2-alkanols, such as 2-tetradecanol and 2-dodecanol, were used as rather unconventional hydrophobic carbon sources. Based on the composition of the sophorolipids, Brakemeier obtained a relative amount of these molecules in the total mixture lower than 7.4% when produced from 2-tetradecanol and lower than 0.5% when produced from 2-dodecanol. In other words, despite the use of non-conventional and expensive substrates, the overall obtained amount of these bolaform molecules was still very low.
Recently, Price et al. (2012) described the production of novel sophorolipids by the species Candida sp. NRRL Y-27208 when grown on a normal production medium (fatty acids as the hydrophobic carbon source). Among the novel sophorolipids, 17 dimeric (=comprising 4 glucose molecules) and trimeric (=comprising 6 glucose molecules) sophoroses were identified. Dimeric molecules were also reported for Starmerella (Candida) bombicola. The authors state that these compounds were produced only in minor amounts, close to the detection limit. The majority of the sophorolipids were the conventional ones as shown in FIG. 1.
Taken together, to date, bolaform glycolipids can only be obtained in minor amounts when using conventional hydrophobic carbon sources. The use of unconventional substrates can only lead to a slight increase of the bolaform moiety. Moreover, the latter process is uneconomical due to the use of the very expensive special substrates. Hence, there is a need to efficiently produce bolaamphiphilic sophorolipids in significant amounts starting from cheap conventional carbon sources.